Eucaryotic cytochrome c oxidase, the terminal complex of the respiratory chain, is located in the mitochondrial inner membrane where it is assembled from both mitochondrially and nuclearly encoded subunits. Biosynthesis of this heteroligomeric complex requires the expression of a large number of nuclear genes whose products catalyze processing of the mitochondrial pre-mRNAs, translation of the mRNAs, and a host of post-translational events. At least a dozen such ancillary factors have been implicated to function at late stages of the assembly process, subsequent to synthesis, import and membrane insertion of all the catalytic and structural subunits. Most of our efforts during the prior grant period have been channeled towards attaining a better understanding of the biochemical lesions in assembly-defective mutants. As a result specific functions were ascribed to three proteins, one in heme A synthesis (Coxl0p) and two in mitochondrial copper homeostasis (Coxl7p, Sco1p). Several other mitochondrial proteins involved at post- translational stages of cytochrome oxidase assembly were also characterized. The present proposal has the following goals. 1) To reconstitute an in vitro system for studying copper transfer from Cox17p to Sco1p and subsequently to the apo-subunit 2 of cytochrome oxidase. These studies will make use of purified soluble Cox17p, liposome-bound Sco1p, and of submitochondrial particles containing unassembled subunit 2. 2) To study the kinetics of assembly of the constituent subunits by pulse-chase experiments. This technique will also be used to probe the stages at which enzyme assembly is blocked in mutants. 3) To screen for mutants in the terminal step of heme A synthesis. 4) To combine genetic and biochemical approaches in elucidating the functions of the gene products represented by the class of assembly-defective strains. Since the mechanism of cytochrome oxidase assembly is anticipated to be similar in all eucaryotes, information gained from these studies should be helpful in future analyses of human disorders stemming from cytochrome oxidase deficiencies.